Biological molecules, such as proteins and nucleic acids are routinely fractionated and characterized, e.g., by capillary electrophoresis or using microfluidic separation technology. For example, U.S. Pat. No. 5,948,227 by Dubrow, entitled “Methods and Systems for Performing Electrophoretic Molecular Separations,” describes methods for electrophoretically separating molecular and macromolecular species in microfluidic devices.
Electrophoretic forces are typically used to separate materials in microfluidic devices, e.g., relying upon the electrophoretic mobility of charged species within an electric field applied to the material. Electrophoretic movement is used to separate mixtures of components as they move through a microfluidic channel. Signal peak area of separated components is typically used to assess the extent of reactions, reaction rate constants, concentration of reactants, products, separated components, and a variety of other chemical and biochemical parameters.
Just as in traditional capillary electrophoresis, electrokinetic sample introduction in a microfluidic device biases sample introduction. The electric fields can cause preferential movement of reagents due to differences in their mass to charge ratio, e.g., highly charged materials move to the front or back of a fluid plug. This effect is desirable when attempting to electrokinetically separate various compounds, but inhibits the ability to obtain measurements relating to entire samples, e.g., unseparated unbiased samples. For example, it is often desirable to identify the total concentration, e.g., of nucleic acids or proteins, in a sample in addition to the concentration of each nucleic acid fragment, e.g., after separation.
The calculation of kinetic constants in flowing systems has also been described. For example, published PCT application WO 98/56956, by Kopf-Sill et al., entitled “Apparatus and Methods for Correcting Variable Velocity in Microfluidic Systems,” describes methods of determining concentration, e.g., after an electrokinetically biased sample introduction, using variable velocities, e.g., of reactants and products. This reference also describes, e.g., the use of gated injections to achieve representative sample aliquots and other related phenomena.
The present invention provides methods and apparatus for obtaining representative or unbiased sample aliquots that are used to determine, e.g., total analyte concentrations. The methods and apparatus of the present invention provide these features and many others that will be apparent upon complete review of the following disclosure.